Antibiogram, biochemical reactions and genotyping characterization of biofield treated staphylococcu

American Journal of BioScience 2015; 3(6): 212-220

Published online October 15, 2015 (http://www.sciencepublishinggroup.com/j/ajbio)

doi: 10.11648/j.ajbio.20150306.13

ISSN: 2330-0159 (Print); ISSN: 2330-0167 (Online)

Antibiogram, Biochemical Reactions and Genotyping Characterization of Biofield Treated Staphylococcus aureus

Mahendra Kumar Trivedi1, Alice Branton

1, Dahryn Trivedi

1, Gopal Nayak

1,

Sambhu Charan Mondal2, Snehasis Jana

2, *

1Trivedi Global Inc., Henderson, USA 2Trivedi Science Research Laboratory Pvt. Ltd., Hall-A, Chinar Mega Mall, Chinar Fortune City, Hoshangabad Rd., Bhopal, Madhya

Pradesh, India

Email address: [email protected] (S. Jana)

To cite this article: Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Sambhu Charan Mondal, Snehasis Jana. Antibiogram, Biochemical

Reactions and Genotyping Characterization of Biofield Treated Staphylococcus aureus. American Journal of BioScience.

Vol. 3, No. 6, 2015, pp. 212-220. doi: 10.11648/j.ajbio.20150306.13

Abstract: Staphylococcus aureus (S. aureus) is the key organism for food poisoning due to massive production of heat stable

exotoxins. The current study was attempted to investigate the effect of Mr. Trivedi’s biofield treatment on S. aureus. S. aureus

(ATCC 25923) was divided into two parts, Group (Gr.) I: control and Gr. II: treatment. After biofield treatment, Gr. II was

further subdivided into two parts, Gr. IIA and Gr. IIB. Gr. IIA was analyzed on day 10, while Gr. IIB was stored and analyzed

on day 159 after revival (Study I). The revived sample (Gr. IIB) were retreated on day 159 (Study II), and divided into three

separate tubes. Tube 1 was analyzed on day 5, likewise, tube 2 and 3 were analyzed on day 10 and 15, respectively. All the

experimental parameters were studied using automated MicroScan Walk-Away® system. The 16S rDNA sequencing was

carried out in Gr. IIA sample to correlate the phylogenetic relationship of S. aureus with other bacterial species. The

antimicrobial susceptibility and minimum inhibitory concentration showed significant alteration i.e. 92.86% and 90.00%

respectively in treated cells of S. aureus as compared to control. The biochemical reactions also showed the significant

(35.71%) alteration in treated sample with respect to control. The biotype number and microbial species were substantially

changed in Gr. IIA (767177; Staphylococcus cohnii subsp. urealyticum) on day 10, while only the biotype numbers were

changed in rest of the treated samples as compared to control (307016; S. aureus). The 16S rDNA analysis showed that the

identified strain in this experiment was S. aureus (GenBank Accession No.: L37597) after biofield treatment. However, the

nearest homolog genus-species was found as Staphylococcus simiae (GenBank Accession No.: DQ127902). These results

suggested that biofield treatment has a significant impact on S. aureus in lyophilized as well as revived state.

Keywords: Staphylococci, Staphylococcus aureus, Antimicrobial Sensitivity, Biofield Treatment, Biochemical Reaction,

Biotype, 16S rDNA, Gram-Positive Bacteria

1. Introduction

Staphylococci are the important class of pyogenic Gram-

positive spherical bacteria resembling to the grapes like

structure. They are considered as the third most important

cause of food-borne disorders in the world [1]. It is the

main pathogen for mastitis in the milch animals [2]. It is

estimated that in US alone food-borne illnesses affect 6 to

80 million people each year, causing up to 9000 deaths [3].

Based on literature various genes have been found as a

target for identification of S. aureus with the help of 16S

rDNA sequence viz. heat shock protein 60 (hsp60) [4],

superoxide dismutase A (sodA) [5], and RNA polymerase B

(rpoB) [6]. S. aureus has developed resistance to the most

classes of the antimicrobial agents. Penicillin is the drug of

choice to treat against Staphylococcus infection but due to

penicillinase or β-lactamase enzyme that destroy the

penicillin, leads to resistance against S. aureus [7].

Therefore, some alternative strategies are needed to treat

against staphylococci infections.

National Institute of Health/National Center for

Complementary and Alternative Medicine (NIH/NCCAM)

have reported that biofield (putative energy fields) or

electromagnetic based energy therapies used to promote

American Journal of BioScience 2015; 3(6): 212-220 213

health and healing [8]. Biofield energy treatment has been

known as an alternative approach that may be useful to alter

the sensitivity pattern of the antimicrobials. Harold Saxton

Burr had performed the detailed studies on the correlation

of electric current with physiological processes and

suggested that every single process in the human body had

an electrical significance [9]. The electrical process that

happening in the human body have strong relationship with

magnetic field as required by Ampere’s law, which stated

that the moving charge produces magnetic field in

surrounding space [10, 11]. Thus, the human body emits the

electromagnetic waves in the form of bio-photons, which

surrounds the body and it is commonly known as biofield.

Therefore, the biofield consists of an electromagnetic field,

being generated by moving electrically charged particles

(ions, cell, molecule, etc.) inside the human body. Prakash

et al. 2015, reported that the various scientific instruments

such as Kirlian photography, polycontrast interference

photography and resonance field imaging can be

extensively used to measure the biofield of human body

[12]. Thus, a human has the ability to harness the energy

from environment or universe and can transmit into any

living or nonliving object(s) around the Globe. The objects

always receive the energy and respond into useful way that

is called biofield energy and the process is known as

biofield treatment. Mr. Trivedi’s biofield treatment (The

Trivedi Effect®

) has been known to alter the structural,

physical and thermal properties of several metals in

materials science [13-15], improved the overall productivity

of crops [16, 17], altered characteristics features of

microbes [18-20] and improved growth and anatomical

characteristics of various medicinal plants [21, 22]. Due to

the clinical significance of this organism and literature

reports on biofield treatment, the present work was

undertaken to evaluate the impact of biofield treatment

modality on S. aureus in relation to the antimicrobials

susceptibility, biochemical reactions, biotyping and 16S

rDNA sequencing.

2. Materials and Methods

S. aureus, American Type Culture Collection (ATCC

25923) strain was procured from MicroBioLogics, Inc.,

USA and stored with proper storage conditions until further

use. All the tested antimicrobials and biochemicals were

procured from Sigma-Aldrich (MA, USA). The

antimicrobial susceptibility, biochemical reactions and

biotype number were estimated with the help of MicroScan

Walk-Away®

(Dade Behring Inc., West Sacramento, CA,

USA) using Positive Breakpoint Combo 20 (PBPC 20)

panel. The 16S rDNA sequencing analysis was carried out

using ultrapure genomic DNA prep kit; Cat KT 83

(Bangalore Genei, India).

2.1. Experimental Design

The impact of biofield treatment on tested bacterium S.

aureus was evaluated in two groups-

Group I: ATCC strain in lyophilized state was considered

as control. No treatment was given and analyzed for

antimicrobial sensitivity, biochemical reactions and biotype

number as per the standard protocol.

Group II: The lyophilized state of ATCC strain was

divided into two parts named as Gr. IIA and Gr. IIB. Both the

groups of ATCC strain of S. aureus in lyophilized state were

assigned to the Mr. Trivedi’s unique biofield treatment (first

treatment). Gr. IIA was analyzed on day 10 while Gr. IIB

sample was stored in lyophilized state for 159 days at -70ºC.

Gr. IIB was further sub-divided in two separate parts named

as Gr. IIB - Study I and Gr. IIB - Study II.

Group IIB - Study I

After 159 days, antimicrobial sensitivity, MIC,

biochemical reactions and biotyping were performed as per

the standard protocol.

Group IIB - Study II

The stored strain was revived from -70ºC and the revived

culture was again provided to Mr. Trivedi’s biofield

treatment (re-treatment) on day 159. After biofield

retreatment, the sample was sub-cultured into three separate

tubes and analyzed on Day 5, Day 10 and Day 15 of its sub-

culturing.

2.2. Biofield Treatment Strategy

The lyophilized sample of S. aureus was subjected to Mr.

Trivedi’s biofield treatment (first treatment) and then

stored, analyzed on day 10 (Gr. IIA) followed by

retreatment on 159 days in revived state (Gr. IIB, Study II).

In details, the treatment groups in sealed pack were handed

over to Mr. Trivedi for biofield treatment under laboratory

conditions. Mr. Trivedi provided the treatment through his

energy transmission process to the treated group without

touching the samples. After first treatment, the analysis of

Gr. IIA lyophilized sample was done on day 10 for

antimicrobial sensitivity along with minimum inhibitory

concentration (MIC), biochemical reactions with biotype

number and 16S rDNA analysis as per the standard

protocol. While handing over these cultures to Mr. Trivedi

for retreatment purposes, optimum precautions were taken

to avoid contamination.

2.3. Antimicrobial Susceptibility Test

Investigation of antimicrobial susceptibility of S. aureus

was carried out with the help of automated instrument,

MicroScan Walk-Away®

using PBPC 20 panel. The panel

can be stored at 2 to 25ºC for analysis. The panel was

allowed to equilibrate to room temperature prior to

rehydration. All opened panels were used on the same day.

The tests carried out on MicroScan were miniaturized of the

broth dilution susceptibility test that has been dehydrated.

Briefly, 0.1 mL of the standardized suspension of S. aureus

was pipetted into 25 mL of inoculum water using pluronic

and inverted 8 to 10 times and inoculated, rehydrated, and

then subjected to incubation for 16 hours at 35°C.

Rehydration and inoculation were performed using the

214 Mahendra Kumar Trivedi et al.: Antibiogram, Biochemical Reactions and Genotyping

Characterization of Biofield Treated Staphylococcus aureus

RENOK®

system with inoculators-D (B1013-4). 25 mL of

standardized inoculum suspension was poured into

inoculum tray. The detailed experimental procedure and

conditions were followed as per the manufacturer's

instructions. The antimicrobial susceptibility pattern (S:

Susceptible, R: Resistant; and BLAC: β-lactamase positive)

and MIC were determined by observing the lowest

antimicrobial concentration showing inhibition of growth

[23].

2.4. Biochemical Reaction Studies

Biochemical reactions of S. aureus were determined

using MicroScan Walk-Away®

, system with PBPC 20 panel.

Preparation of PBPC 20 panel, inoculum followed by

dehydration and rehydration were performed in a similar

way as mentioned in antimicrobial susceptibility assay for

analysis of biochemical reactions followed by biotype

number. The detailed experimental procedures and

conditions were followed as per the manufacturer's

instructions [23].

2.5. Identification of Organism by Biotype Number

The biotype number of S. aureus was determined on

MicroScan Walk-Away® processed panel data report with the

help of biochemical reactions data [23, 24].

2.6. Amplification and Gene Sequencing of 16S rDNA

Genomic DNA was isolated from S. aureus cells (Gr. IIA,

sample coded as 9A) using genomic purification kit, according

to the manufacturer instructions. 16S rDNA gene (~1.5 kb)

fragment was amplified with the help of high-fidelity

polymerase chain reaction (PCR) using universal primers;

forward primer (5ˊ-AGAGTTTGATCCTGGCTCAG-3ˊ) and

reverse primer (3ˊ-ACGGTCATACCTTGTTACGACTT-5ˊ).

Amplified products were subjected to gel electrophoresis in

1.0% agarose gel, stained with ethidium bromide and

visualized under UV light in a gel documentation unit (BioRad

Laboratories, USA). The PCR amplified fragment was purified

from the agarose gel using a DNA gel extraction kit.

Sequencing of amplified product was done on a commercial

basis from Bangalore Genei, India. The 16S rDNA sequences

obtained were aligned and compared with the sequences stored

in GenBank database available from National Center for

Biotechnology Information (NCBI) using the algorithm

BLASTn program. Multiple sequence alignment /

phylogenetic tree were established using MEGA3.1 molecular

software [25].

3. Results and Discussion

3.1. Antimicrobial Susceptibility Test

The results of S. aureus susceptibility pattern and MIC

values of tested antimicrobials after biofield treatment are

summarized in Table 1 and 2 respectively. The data were

analyzed and compared with respect to control.

Antimicrobial sensitivity assay and MIC were performed in

twenty-eight and thirty antimicrobials respectively. The

treated cells of S. aureus showed a significant (85.71%)

alteration (twenty-four out of twenty-eight) in antimicrobial

sensitivity pattern from susceptible (S) to resistance (R) in

lyophilized treated Gr. IIA on day 10 after first-time

biofield treatment as compared with control. However,

these twenty-four antimicrobials did not show any change

of sensitivity pattern on day 159 as well as in revived state

even after retreatment as compared to control. Out of

twenty-eight antimicrobials two antibiotics i.e. ampicillin

and penicillin were changed from S to β-lactamase positive

(BLAC) in lyophilized treated Gr. IIA on day 10 while

showed similar response in rest of treated groups even after

second-time biofield treatment as compared with control. S.

aureus has the ability to produce β-lactamases or

penicillinase enzyme which breakdown the β-lactam ring

present in penems and cephems heteronucleus [26]. Two,

out of twenty eight (7.14%) tested antimicrobials such as

piperacillin/tazobactam and linezolid did not show any

responses in lyophilized treated cells (Gr. IIA) on day 10

while they exhibited susceptible in rest of treated samples

of S. aureus. Overall, 92.86% antimicrobial susceptibility

pattern was altered after biofield treatment as compared to

control. MIC values of several antimicrobials viz.

ampicillin/sulbactam, azithromycin, cefazolin, cefepime,

cephalothin, chloramphenicol, ciprofloxacin, gatifloxacin,

gentamicin, levofloxacin, linezolid, moxifloxacin,

norfloxacin, ofloxacillin, rifampin, tetracycline, and

synercid showed an alteration about two-fold in Gr. IIA on

day 10 as compared to control. The MIC value of

cefotaxime, ceftriaxone and clindamycin were changed

about four-fold in Gr. IIA on day 10 while remained

unchanged in rest of the groups as compared to control.

Certain antimicrobials such as erythromycin, oxacillin and

vancomycin showed eight-fold, while ampicillin showed

thirty two-fold (≤0.25 to >8 µg/mL) and penicillin showed

around two hundred sixty seven-fold (≤0.03 to >8 µg/mL)

alteration of MIC values in Gr. IIA on day 10 as compared to

control. Amoxicillin / k-clavulanate and trimethoprim /

sulfamethoxazole were slightly altered the MIC values in Gr.

IIA on day 10. Overall, 90% out of thirty tested

antimicrobials showed an alteration of MIC values as

compared to control. Three out of thirty (10%) antimicrobials

did not show any alteration of MIC values in all the treated

groups as compared to control (Table 2) except piperacillin /

tazobactam in Gr. IIA, value not reported. Overall, the

antimicrobial resistance pattern (S to R) and corresponding

MIC values were significantly altered in lyophilized strain S.

aureus after first-time biofield treatment as compared to

control.

3.2. Biochemical Reactions Studies

Data obtained from biochemical reactions studies for

distinction of S. aureus are illustrated in Table 3. Study of

biochemical reactions can be utilized to identify the

enzymatic and metabolic characteristic feature of microbes.


Microorganisms can be categorically differentiated based

on their utilization of specific biochemicals as nutrients

during the process of metabolism or enzymatic reactions.

Biochemicals such as arginine (ARG), mannose (MNS) and

urea (URE) were changed from negative (-) to positive (+)

reactions in all the treated groups with respect to control.

Moreover, biochemical reactions of β-lactamases (BL),

crystal violet (CV), novobiocin (NOV), galactosidase (PGR

and PGT) and sorbitol (SOR) were converted from negative

(-) to positive (+) reactions in Gr. IIA on day 10 after first

biofield treatment and remained same i.e. negative (-) in

rest of the treated samples as compared to control. The

alterations of biochemical reactions after biofield treatment

of ARG, PGR and PGT were the typical characteristics

feature of S. aureus. Based on this data, it is assumed that

Mr. Trivedi’s biofield treatment has an impact on S. aureus

in term of metabolic reaction. Similarly, rambose (RBS)

was converted from negative (-) to the positive (+) reaction

after first-time biofield treatment in Gr. IIA on day 10 and

after retreatment in Gr. IIB, Study II on day 15 while

remained unchanged in rest of the treated groups as

compared to control. The key characteristic feature for S.

aureus are colony pigment, free coagulase, clumping factor,

protein A, heat-stable nuclease and acid production from

mannitol [27]. In this experiment, due to the production of

acid from mannitol, the result showed positive (+) reaction

in all the treated groups which supports the metabolic

characteristics feature of S. aureus. Overall, 35.71%

biochemical reactions were altered in tested twenty-eight

biochemicals with respect to control after biofield

treatment. About 64.29% of total biochemicals, such as

arabinose, bacillosamine, bile esculin, indoxyl phosphatase,

inulin, acidification lactose, mannitol, micrococcus screen,

sodium chloride, nitrate, optochin, phosphatase, pyruvate,

pyrolidonyl arylamidase, raffinose, thymidine free growth,

acidification trehalose and Voges-Proskauer did not show

any change in all the groups after biofield treatment as

compared to control.

Table 1. Antibiogram of Staphylococcus aureus: effect of biofield treatment on antimicrobial susceptibility.

S. No. Antimicrobial Gr. I (Control) Gr. IIA (Day 10) Gr. IIB (Study I;

Day 159)

Gr. IIB (Study II; Day 159)

Day 5 Day 10 Day 15

1. Amoxicillin/k-clavulanate S R S S S S

2. Ampicillin/sulbactam S R S S S S

3. Ampicillin S BLAC S S S S

4. Azithromycin S R S S S S

5. Cefazolin S R S S S S

6. Cefepime S R S S S S

7. Cefotaxime S R S S S S

8. Ceftriaxone S R S S S S

9. Cephalothin S R S S S S

10. Chloramphenicol S R S S S S

11. Ciprofloxacin S R S S S S

12. Clindamycin S R S S S S

13. Erythromycin S R S S S S

14. Gatifloxacin S R S S S S

15. Gentamicin S R S S S S

16. Imipenem S R S S S S

17. Levofloxacin S R S S S S

18. Linezolid S NA S S S S

19. Moxifloxacin S R S S S S

20. Ofloxacillin S R S S S S

21. Oxacillin S R S S S S

22. Penicillin S BLAC S S S S

23. Piperacillin/tazobactam S − S S S S

24. Rifampin S R S S S S

25. Synercid S R S S S S

26. Tetracycline S R S S S S

27. Trimethoprim/sulfamethoxazole S R S S S S

28. Vancomycin S R S S S S

R: Resistant; S: Susceptible; Gr.: Group; BLAC: β-lactamase positive; NA: Data not available;

-, Not reported



Table 2. Effect of biofield treatment on Staphylococcus aureus to minimum inhibitory concentration (MIC) of tested antimicrobials.

S. No. Antimicrobial Gr. I (Control) Gr. IIA (Day 10) Gr. IIB (Study I;

Day 159)


Day 5 Day 10 Day 15

1. Amoxicillin/k-clavulanate ≤4/2 ˃4/2 ≤4/2 ≤4/2 ≤4/2 ≤4/2

2. Ampicillin/sulbactam ≤8/4 ˃16/8 ≤8/4 ≤8/4 ≤8/4 ≤8/4

3. Ampicillin ≤0.25 ˃8 ≤0.25 ≤0.25 ≤0.25 ≤0.25

4. Azithromycin ≤2 ˃4 ≤2 ≤2 ≤2 ≤2

5. Cefazolin ≤8 ˃16 ≤8 ≤8 ≤8 ≤8

6. Cefepime ≤8 ˃16 ≤8 ≤8 ≤8 ≤8

7. Cefotaxime ≤8 ˃32 ≤8 ≤8 ≤8 ≤8

8. Ceftriaxone ≤8 ˃32 ≤8 ≤8 ≤8 ≤8

9. Cephalothin ≤8 ˃16 ≤8 ≤8 ≤8 ≤8

10. Chloramphenicol ≤8 ˃16 ≤8 ≤8 ≤8 ≤8

11. Ciprofloxacin ≤1 ˃2 ≤1 ≤1 ≤1 ≤1

12. Clindamycin ≤0.5 ˃2 ≤0.5 ≤0.5 ≤0.5 ≤0.5

13. Erythromycin ≤0.5 ˃4 ≤0.5 ≤0.5 ≤0.5 ≤0.5

14. Gatifloxacin ≤2 ˃4 ≤2 ≤2 ≤2 ≤2

15. Gentamicin ≤4 ˃8 ≤4 ≤4 ≤4 ≤4

16. Imipenem ≤4 ≤4 ≤4 ≤4 ≤4 ≤4

17. Levofloxacin ≤2 ˃4 ≤2 ≤2 ≤2 ≤2

18. Linezolid ≤2 ˃4 ≤2 ≤2 ≤2 ≤2

19. Moxifloxacin ≤2 ˃4 ≤2 ≤2 ≤2 ≤2

20. Nitrofurantoin ≤32 ≤32 ≤32 ≤32 ≤32 ≤32

21. Norfloxacin ≤4 ˃8 ≤4 ≤4 ≤4 ≤4

22. Ofloxacillin ≤2 ˃4 ≤2 ≤2 ≤2 ≤2

23. Oxacillin ≤0.25 ˃2 ≤0.25 ≤0.25 ≤0.25 ≤0.25

24. Penicillin ≤0.03 ˃8 ≤0.03 ≤0.03 ≤0.03 ≤0.03

25. Piperacillin/tazobactam ≤4 − ≤4 ≤4 ≤4 ≤4

26. Rifampin ≤1 ˃2 ≤1 ≤1 ≤1 ≤1

27. Synercid ≤1 ˃2 ≤1 ≤1 ≤1 ≤1

28. Tetracycline ≤4 ˃8 ≤4 ≤4 ≤4 ≤4

29. Trimethoprim/sulfamethoxazole ≤2/38 ˃2/38 ≤2/38 ≤2/38 ≤2/38 ≤2/38

30. Vancomycin ≤2 ˃16 ≤2 ≤2 ≤2 ≤2

MIC data are presented in µg/mL; Gr.: Group; -, Not reported

Table 3. Effect of biofield treatment on Staphylococcus aureus to the biochemical reaction pattern.

S. No. Code Biochemical Gr. I (Control) Gr. IIA

(Day 10)

Gr. IIB (Study I;

Day 159)


Day 5 Day 10 Day 15

1. ARA Arabinose - - - - - -

2. ARG Arginine - + + + + +

3. BAC Bacillosamine + + + + + +

4. BE Bile esculin - - - - - -

5. BL β-lactamases - + - - - -

6. CV Crystal violet - + - - - -

7. IDX Indoxyl phosphatase - - - - - -

8. INU Inulin - - - - - -

9. LAC Acidification Lactose + + + + + +

10. MAN Mannitol + + + + + +

11. MNS Mannose - + + + + +

12. MS Micrococcus screen + + + + + +

13. NACL Sodium chloride + + + + + +

14. NIT Nitrate + + + + + +

15. NOV Novobiocin - + - - - -

16. OPT Optochin + + + + + +

17. PGR Glycosidase* - + - - - -

18. PGT Glycosidase# - + - - - -

19. PHO Phosphatase + + + + + +

20. PRV Pyruvate - - - - - -

21. PYR Pyrolidonyl arylamidase - - - - - -

22. RAF Raffinose - - - - - -

23. RBS Rambose - + - - - +

24. SOR Sorbitol - + - - - -

25. TFG Thymidine free growth + + + + + +

26. TRE Acidification trehalose + + + + + +

27. URE Urea - + + + + +

28. VP Voges-Proskauer + + + + + +

-, (negative); +, (positive); Gr.: Group; *PGR: p-nitro phenyl β-D- glucuronide; #PGT: p-nitro phenyl β-D-galactopyranoside


3.3. Identification of Organism by Biotype Number

The species (S. aureus) was identified based on variety of

conventional biochemical characters and biotyping. Biotype

number of particular organism was evaluated after

interpreting the results of the biochemical reactions. The

biotype number then led to the particular organism

identification. In this experiment, biotyping was performed

using an automated system, and results showed a significant

change in biotype number (767177) in Gr. IIA (on day 10)

after first-time biofield treatment with identification of new

species (Staphylococcus cohnii subsp. urealyticum) as

compared to control Gr. I (307016; S. aureus). Based on the

biochemical results, biotype numbers were also changed in

rest of treated groups without alteration of species with

respect to control (307016) i.e. S. aureus (Table 4).

Table 4. Effect of biofield treatment on biotype number of Staphylococcus aureus.

Feature Gr. I (Control) Gr. IIA (Day 10) Gr. IIB (Study I; Day 159) Gr. IIB (Study II; Day 159)

Day 5 Day 10 Day 15

Biotype 307016 767177 307137 307137 307137 307137

Organism Identification S. aureus Staphylococcus cohnii subsp. urealyticum S. aureus S. aureus S. aureus S. aureus

Gr.: Group

3.4. 16S rDNA Genotyping

The bacteria that are poorly differentiated by

conventional methods needs molecular analysis method like

16S rDNA sequence [28]. This molecular-based technique

is a suitable tool for identification of most of the bacteria on

their genus and/or species level by comparison with

databases in the public domain. Because, most of the

bacteria possess small ribosomal subunit with species-

specific variability [29]. The 16S rDNA analysis was

performed after first-time biofield treated sample (Gr. IIA)

of S. aureus on day 10. The alignment and comparison of

the consensus gene sequences were performed with the

sequences stored in GenBank database available from

NCBI using the algorithm BLASTn program. Based on

nucleotide homology and phylogenetic analysis the microbe

(Sample 9A) was detected as Staphylococcus aureus

(GenBank Accession No: L37597) with 100% identity. The

nearest homolog genus-species of S. aureus was found as

Staphylococcus simiae (GenBank Accession No.

DQ127902). Some other close homologs of S. aureus were

found from the alignment results as shown in Table 5. The

distance matrix based on nucleotide sequence homology

data are presented in Table 6. The phylogenetic tree was

established using BLAST-Webpage (NCBI). According to

Table 6, ten different related bacterial species of S. aureus

were selected as Operational Taxonomic Units (OTUs) in

order to investigate the phylogenetic relationship of S.

aureus. There were 1497 base nucleotides of 16S rDNA

gene sequences, which were analyzed and multiple

alignments were constructed using ClustalW in MEGA3.1.

The numbers of base substitutions per site from pairwise

distance analysis between sequences are shown in Table 5.

All results were based on the pairwise analysis of 11

sequences. According to the data in Table 6, the lowest

value of the genetic distance from S. aureus was 0.000 base

substitutions per site. This value is due to the distance

between S. aureus and Staphylococcus simiae. All pairwise

distance analysis was carried out using the p-distance

method in MEGA3.1. The proportion of remarked distance,

sometimes also called p-distance and showed as the number

of nucleotide distances site. Values in Table 5 are

programmed into Figure 1 with optimal bootstrap consensus

tree. In the phylogram, there were eleven OTUs. The results

suggested that S. aureus was closely related to the

Staphylococcus simiae with 100% similarity and the lowest

genetic distance 0.000 base substitutions per site.

Table 5. The closest sequences of Staphylococcus aureus from sequence

alignment using NCBI GenBank and ribosomal database project (RDP).

Alignment View AN Alignment

Result Sequence Description

9A 1.00 Sample studied

CP000730 1.00 Staphylococcus aureus

subsp. aureus USA300

AP009324 1.00 Staphylococcus aureus

subsp. aureus Mu3

CP000736 1.00 Staphylococcus aureus

subsp. aureus JH1

AB353073 1.00 Staphylococcus aureus

strain: MPU99

L37597 1.00 Staphylococcus aureus

DQ997837 1.00 Staphylococcus aureus

strain ATCC 14458


strain ATCC 14458


isolation-source bhalla

DQ127902 0.97 Staphylococcus simiae

strain CCM 7229

EF522128 0.99

Staphylococcus

epidermidis strain

CU22

AN: GenBank Accession Number



Table 6. Distance matrix Staphylococcus aureus sample (9A) based on nucleotide sequence homology (Using Kimura-2 Parameter) indicates nucleotide

similarity (above diagonal) and distance (below diagonal) identities between the studied sample ‘9A’ and ten closest homologs microbe.

Distance Matrix

AN

1 2 3 4 5 6 7 8 9 10 11

CP000736 1 — 1 1 0.998 1 0.998 1 0.995 0.986 1 1

DQ997837 2 0.000 — 1 0.998 1 0.998 1 0.995 0.986 1 1

AP009324 3 0.000 0.000 — 0.998 1 0.998 1 0.995 0.986 1 1

DQ269498 4 0.002 0.002 0.002 — 0.998 0.999 0.998 0.995 0.984 0.998 0.998

AB353073 5 0.000 0.000 0.000 0.002 — 0.998 1 0.995 0.986 1 1

DQ630752 6 0.002 0.002 0.002 0.001 0.002 — 0.998 0.995 0.984 0.998 0.998

CP000730 7 0.000 0.000 0.000 0.002 0.000 0.002 — 0.995 0.986 1 1

DQ127902 8 0.005 0.005 0.005 0.005 0.005 0.005 0.005 — 0.985 0.995 0.995

EF522128 9 0.014 0.014 0.014 0.016 0.014 0.016 0.014 0.015 — 0.986 0.986

L37597 10 0.000 0.000 0.000 0.002 0.000 0.002 0.000 0.005 0.014 — 1

9A 11 0.000 0.000 0.000 0.002 0.000 0.002 0.000 0.005 0.014 0.000 —

AN: GenBank Accession Number

Numbers represent GenBank accession number

Figure 1. Phylogenetic tree of the partial 16S rDNA gene sequencing of Staphylococcus aureus using MEGA 3.1 software using neighbor joining method.

Numbers represent GenBank accession number.

Biofield treatment may be responsible for alteration in

microorganism at enzymatic and/or genetic level, which may

act on receptor protein. While altering receptor protein,

ligand-receptor/protein interactions may altered that could

lead to show different phenotypic characteristics [30].

Biofield treatment might induce significant changes in

lyophilized strain of S. aureus and altered antimicrobials

susceptibility pattern, MIC values, biochemical reactions,

which ultimately change the biotype number of

microorganism. As a result, the microbe that was susceptible

to a particular antimicrobial in control sample now converted

into BLAC/resistant in treated cells of S. aureus

predominately after biofield treatment. Due to microbial

resistance to a single drug or multiple drugs, the invention of

an effective antimicrobial therapy for the human-wellness is

urgently required. However, due to some limitation of

science, the progress of new medications is slow and very

challenging for scientists. So far our group had published

many research articles regrading short-term effects on

biofield treatment on ATCC and multidrug resistant strains

[18-20]. Based on these results, it is envisaged that biofield

treatment has the ability to alter the sensitivity pattern of

antimicrobials and scope to be an alternative approach than

the existing antimicrobial therapy in near future.

4. Conclusions

In conclusion, the antimicrobial susceptibility pattern and

the MIC values showed the significant 92.86% and 90%

alteration, respectively of tested antimicrobials as compared


to the control strain of S. aureus. The biochemical reactions

pattern showed the significant 35.71% alteration as compared

to the control. Moreover, the biotype numbers of biofield

treated strain of S. aureus were also changed in all the treated

groups as compared to the control. Based on the changed

biotype numbers after biofield treatment, new species was

identified as (767177; Staphylococcus cohnii subsp.

urealyticum) in lyophilized treated cells (Gr. IIA) on day 10

with respect to the control Gr. I (307016; S. aureus). Thus,

Mr. Trivedi’s unique biofield energy treatment could be

applied as an alternative therapeutic approach against

antimicrobials to alter the sensitivity pattern. Molecular

based 16S rDNA analysis showed that the treated lyophilized

sample in this experiment was S. aureus. However, the

nearest homolog genus-species was found to be

Staphylococcus simiae. Based on these results, it seems that

biofield treatment could be used as an alternate of existing

drug therapy in future.

Acknowledgment

Authors gratefully acknowledged to Trivedi science,

Trivedi testimonials and Trivedi master wellness and the

whole team of PD Hinduja National Hospital and MRC,

Mumbai, Microbiology Lab for their support. Authors also

would like thanks Bangalore Genei Pvt. Ltd., for conducting

16S rDNA sequencing analysis.

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Antibiogram, biochemical reactions and genotyping characterization of biofield treated staphylococcu